1. ABSTRACT / SUMMARY We propose to continue our SBIR phase I work and further develop a broadly neutralizing, possibly universal influenza vaccine composed of virus-like particles (VLPs) displaying remodeled HA molecules which exhibit otherwise cryptic epitopes. These remodeled HAs will be expressed recombinantly in forms lacking or shedding the dominant hypervariable epitopes and instead display distinctly conserved subdominant antigenic sites known to promote an antibody response that will neutralize a broad spectrum of influenza viruses. Additionally, the VLP structure displaying the remodeled HA and NA contains more conserved influenza antigens like matrices M1, M2 and the nucleoprotein NP. The protective scope of current influenza vaccines is restricted to homologous viruses or closely related variants and vaccine efficacy wanes following the fast antigenic evolution of the influenza virus. Most protective antibodies target highly variable and dominant sites on the globular head of the HA molecule, although more conserved and less immune-recognized conformational antigenic sites are also present in the stem (HA2) and between the globular head (HA1) and stem portions of HA. Isolated human antibodies directed toward these sites have been found to neutralize a broad spectrum of influenza viruses. It seems reasonable therefore to prepare and test vaccines that display these highly conserved subdominant antigenic sites together with other important influenza antigens and determine if they stimulate a broad antibody response which is minimal in a natural influenza infection or following vaccination with formulations containing whole HA molecules. Incorporation of remodeled HA molecules into influenza virus-like particles (VLPs) should provide an excellent opportunity to develop a broadly neutralizing vaccine. VLPs are generated by the co-expression of five structural influenza proteins (M1, M2, HA, NA and NP) and do not contain infectious viral genetic material and are therefore unable to replicate or cause infection. Studies performed during the phase I SBIR showed that VLP generated with remodeled HA not only elicit neutralizing antibodies but also afford significant protection against a lethal influenza challenge after a single immunization. To continue this work, we propose to further evaluate the VLP based vaccine containing alternative conformations of the remodeled HA together with native or modified NA as surface component of a M1, M2 and NP influenza virus-like particle assembly. This vaccine is produced in suspension culture of mammalian cells suitable for the most advanced fermentation technology and amenable for stable cells line development for continuous manufacturing. This vaccine composition and configuration will elicit humoral neutralizing immunity as well as cell- mediated immunity (CMI) toward the M1 and NP antigens. The immunogenicity and efficacy of the VLP vaccine compositions will be tested in mice and ferrets using as challenge influenza virus subtypes that represent group 1 and group 2 Influenza A virus. A similar strategy will be pursued with the two lineages of influenza B viruses. These studies will provide the foundation to advance development of an influenza vaccine able not only to broaden and enhanced the spectrum of protection but also extend the duration on immunity for several years. Influenza is a constant threat and the current vaccine technology to prevent seasonal or potential pandemic outbreaks is in a permanent catch up mode not only to produce vaccine on time but also to correctly match the antigenic composition of prevailing circulating virus. Even in the best of circumstances the overall effectiveness of the influenza vaccine may range from 40% to 70%. To change this paradigm, we need a vaccine technology that overcomes and withstands antigenic variation and prolongs the duration of induced immunity as well as utilizes rapid, efficient and cost-effective vaccine manufacturing methods. This proposal addresses these issues aiming to advance the development of the vaccine technology and production methods to overcome the limitation of the current vaccine and its production.